Oral treatment of hemophilia

ABSTRACT

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a subject a therapeutically effective amount of the deficient antigen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac a therapeutically effective amount of the appropriate clotting factor other than in a liposome, sufficient to induce oral tolerance and supply exogenous clotting factor to the subject.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional applicationNo. 60/310,150, filed Aug. 3, 2001, which is hereby incorporated byreference in its entirety.

FIELD

[0002] This disclosure relates to methods for treating a subjectsuffering from an antigen-deficiency which causes a disease, by orallyadministering a therapeutically effective amount of the deficientantigen to the subject, sufficient to induce oral tolerance and relievesymptoms of the disease. In one example, the disclosure relates to oraladministration of clotting factor VIII or IX for treating hemophilia Aand B, respectively.

BACKGROUND

[0003] Hemophilia A and B, caused respectively by decreased levels ofclotting factor VIII (F.VIII) and IX (F.IX) levels in peripheral blood,are the most common severe inherited bleeding disorders. Althoughpurified clotting factors from human plasma can be infused into thesepatients to prevent or treat bleeding episodes, this poses the risk ofspreading of diseases such as Creutzfled-Jakob disease, HIV, andhepatitis C. Although recombinant clotting factor preparations areavailable, the supply is insufficient to cover the world-wide demand.

[0004] As an alternative to generating recombinant clotting factors incell culture, the use of transgenic animals as bioreactors for theproduction of clinically useful quantities of proteins has beenproposed. The transgenic animal expresses the desired protein in a bodyfluid, such as milk, from which the protein can readily be isolated.Transgenic pigs that secrete F.VIII (U.S. Pat. No. 5,880,327 to Lubon etal.) or F.IX (Van Cott et al. 1999. Genet. Anal. 15:155-60) in theirmilk were generated using gene constructs that have regulatory sequencesfrom the gene for mouse whey acidic protein (the 4.2 kb 5′ promoter ofWAP), in combination with the cDNA sequence encoding human F.VIII orF.IX. F.VIII pigs secrete 2.7 μg/ml of human F.VIII into milk (Paleyandaet al. 1997. Nat. Biotech. 15:971), while F.IX pigs secrete 2.5 μg/L ofhuman F.IX into milk, and the factor is biologically active as measuredby an activated partial thromboplastin time assay (APTT).

[0005] Therefore, large amounts of functional protein can be obtainedfrom F.IX pigs, which is purified from the milk, and subsequentlyadministered intravenously (i.v.) to a hemophiliac patient. However, ithas previously been thought that oral administration of F.VIII or F.IXresults in degradation and/or inactivation of the protein due to theacidic and enzymatic composition of the stomach. For example, U.S. Pat.No. 4,348,384 to Horikoshi et al. teaches the administration of an oralcomposition containing purified F.VIII or F.IX encapsulated in aliposome along with a protease inhibitor, for the purpose of evadinggastric inactivation of the therapeutic protein.

[0006] One complication that results from i.v. administration of F.VIIIor F.IX is that up to 25% of hemophiliacs develop inhibitors, such asantibodies against F.VIII or F.IX that inactivate their procoagulantactivity (Fields et al. 2000. Mol. Ther. 3:225-35; Bristol et al. 2001.Hum. Gene Ther. 12:1651-61; Ge et al. 2001. Blood 97:3733-7; Brinkhouset al. 1996. Blood 88:2603-10). Most inhibitory antibodies develop inseverely affected patients who have little or no circulating F.IX orF.VIII antigen due to genetic deletion. However, patients with a familyhistory of inhibitor development, severe disease, older age or highernumbers of clotting factor replacement episodes, also have a higherincidence of developing these inhibitors (Roberts. 1997. Inhibitors andtheir management. In Hemophilia & other inherited bleeding disorders.Rizza & Lowe, eds. W B Saunders Company Ltd., London, p. 365.). Theinhibitors can completely inhibit the activity of infused clottingfactor and make further treatment difficult.

[0007] One means of reducing clotting factor antibodies is the inductionof immune tolerance to the clotting factors. For example, Roberts (JAMA259:84-5, 1988) reviews studies in which F.VIII was injected at highconcentrations. Although some studies reported that the level ofanti-factor VIII antibodies decreased, due to the high doses required,the method was never widely used because it was prohibitively expensive.Oral administration of F.VIII as a means to prevent the formation ofanti-F.VIII antibodies has also been attempted, with mixed results. Oraladministration of purified F.VIII to newborn mice did not suppressinduction of anti-F.VIII antibodies (Kaplan et al. 2000. Semin. Thromb.Hemost. 26:173-8). Oral administration of purified F.VIII to one ofthree patients with acquired hemophilia did reduce the amount ofanti-F.VIII antibodies (Lindgren et al. 2000. Thromb. Haemost.83:632-3). However, there are no teachings that oral administration ofclotting factors alone can be used to treat hemophilia which is causedby inadequate expression of a clotting factor.

[0008] Attempts to induce stable production of the missing clottingfactors by gene therapy using injections of transfected myoblasts havealso been hampered by the host immune system which, not being tolerantof the missing factor, generates a strong rejection response whenintroduced (Fields et al. 2000. Mol. Ther. 3:225-35). Thus, inhibitoryantibodies to the factors and vigorous T cell responses to thegenetically transfected cells are major hurdles to successful treatmentof hemophilia.

[0009] Another disadvantage to introduction of a foreign gene throughthe use of a viral vector, is the possibility of an elicited immuneresponse against the vector and/or the transgene product For example,intramuscular (i.m.) injection of an adenoviral vector expressing humanF.IX into the hind limbs of hemophiliac mice results in a CTL responseagainst F.IX, and destruction of the transduced cells, whereas i.m.injection of a less immunogenic vector (adeno-associated virus, AAV)expressing human F.IX results in long-term persistence of the transducedcells and an absence of CTL to F.IX (Roberts. 1997. Inhibitors and theirmanagement. In Hemophilia & other inherited bleeding disorders. Rizza &Lowe, eds., W B Saunders Company Ltd., London, p. 365.). However, inboth cases formation of antibodies neutralizes F.IX activity. The sameproblem exists in the canine model of hemophilia B, where anti-F.IXantibodies neutralize F.IX activity after gene therapy with F.IX (Evanset al. 1989. Proc. Natl. Acad. Sci. U S A. 86:10095-9; Mauser et al.1996. Blood. 88:3451-5; Herzog et al. 1999. Nat. Med. 5:56-63; and Kayet al. 2000. Nat. Genet. 24:257-261).

SUMMARY OF THE DISCLOSURE

[0010] Although potentially large amounts of functional protein can beobtained from F.IX pigs, the current teaching has been to purify thefactor from the milk, and subsequently administer it intravenously(i.v.) to a hemophiliac patient. However, it would be advantageous ifthe protein could be delivered orally, as oral administration is moreeasily handled than injection, and may eliminate the need for proteinpurification. In addition, it would be beneficial if oral tolerance toclotting factors could be induced by oral administration of the clottingfactor.

[0011] Disclosed herein is a method for treating an antigen-deficiencydisease by orally administering a therapeutically effective amount ofthe deficient antigen. In one non-limiting example, the method is amethod for increasing hemostasis in a subject having a hemophilia causedby inadequate expression of a clotting factor, by orally administering atherapeutically effective amount of a clotting factor other than in aliposome. In another example, the method is a method for decreasing thesymptoms associated with Gaucher disease (such as decreasinghepatosplenomegaly and improving bone marrow involvement), in a subjecthaving Gaucher disease caused by decreased expression of acidbeta-glucosidase, by orally administering a therapeutically effectiveamount of a acid beta-glucosidase.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1A is a dot-plot comparing the footpad swelling in mice fedOVA prior to injection (0.1-10 mg OVA) with control mice not fed OVAprior to injection (0 mg OVA).

[0013]FIG. 1B shows three dot-plots comparing the amount of IL-4, TGF-β,and IFN-γ production in mice fed OVA prior to injection, with controlmice not fed OVA (water) prior to injection.

[0014]FIG. 1C is a graph comparing the amount of T-cell proliferation inmice fed OVA prior to injection (0.001-10 mg OVA) with control mice notfed OVA prior to injection (0 mg OVA).

[0015]FIGS. 2A and 2B are graphs comparing the amount of T-cellproliferation in (A) normal and (B) hemophilia B mice fed human F.IXprior to injection, with control mice not fed human F.IX prior toinjection.

[0016]FIG. 2C is a graph comparing the level of IgA produced in F.IX-fedhemophilia B mice to unfed normal mice.

[0017]FIG. 3A and 3B are dot plots showing (A) IgG1 and (B) IgG2aantibody responses to hF.IX.

[0018]FIG. 4 is a dot plot showing that orally tolerized hemophilia Bmice show little evidence of inhibitors, as their bleeding times areonly slightly longer than those of infused mice that had not beenimmunized.

[0019]FIGS. 5A and 5B are graphs comparing the proliferative responseagainst sheep casein, in lambs fed (A) sheep milk or (B) cow milk.

[0020]FIG. 5C is a dot plot comparing the serum IgG titer to sheepcasein in lambs raised on sheep milk compared to cow milk.

[0021]FIGS. 6A and 6B are a graph and a dot plot, respectively, showingthat oral feeding of transgenic pig milk containing F.IX results in thepresence of F.IX in the blood.

[0022]FIG. 7A is a dot plot showing that orally administered F.IXremains functional, and corrects the bleeding defect in hemophilia Bmice.

[0023]FIG. 7B is a graph showing that orally administered F.IX remainsfunctional, and corrects the bleeding defect in hemophilia B mice fedF.IX milk for 2 months.

[0024]FIG. 8A and 8B are dot plots showing that orally administeredF.VIII remains functional, whether administered in water or in cow'smilk.

[0025]FIG. 8C is a dot plot showing that immunization following oraladministration of F.VIII does not inhibit F.VIII fed subsequent to theimmunization.

[0026]FIG. 8D is a dot plot showing IgG1 and IgG2a antibody responses tohF.VIII.

[0027]FIGS. 9A and 9B are graphs comparing the T cell response in micefed F.IX in water or milk showing that oral administration of F.IX canbe achieved whether administered in water or in milk, but that it ismore effective if given in milk.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

[0028] The following explanations of terms and methods are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein and in the appended claims, the singular forms “a” or “an” or“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “a protein” includes a plurality ofsuch proteins and reference to “the antibody” includes reference to oneor more antibodies and equivalents thereof known to those skilled in theart, and so forth.

[0029] Unless explained otherwise, all technical and scientific termsused herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs.

[0030] Antigen: A substance capable of being the target of inducing aspecific immune response.

[0031] Antigen-Deficiency Disease: A disease in which a subject has anantigen deficiency, such as a protein, which causes the disease.Examples of antigen-deficiency diseases include, but are not limited toclotting disorders such as hemophilia A and B (caused by a deficiency ofclotting factor VIII and IX, respectively), Von Willebrand's disease(caused by a moderate-to-severe factor VIII deficiency, low-levels offactor VIII-related antigen, and insufficient von Willebrand factor);and diabetes (caused by a deficiency of insulin).

[0032] Other examples of antigen deficiency diseases include, but arenot limited to: strokes or heart attacks (deficiency of tissueplasminogen activator); emphysema (deficiency of alpha-1-antitrypsin);Gaucher disease (deficiency of β-glucosidase), Pompe's disease(deficiency of alpha-1,4-glucosidase); purpura fulminans neonatalis,warfin-induced skin necrosis, heparin-induced thrombocytopenia, septicshock, and for fibrinolytic therapy (deficiency of protein C) (also seeU.S. Pat. 5,589,604).

[0033] In a particular embodiment, the treatment of such diseases can beachieved by orally administering the deficient antigen to a subjecthaving the disease.

[0034] Anti-immune therapy: Administration of agents to a subject havingan autoimmune disease to reduce the immune response against normal bodytissue while leaving intact the immune response against micro-organismsand abnormal tissues. Examples of agents used to reduce the immuneresponse include, but are not limited to: corticosteroids andimmunosuppressant medications (including prednisolone, cyclophosphamideor azathioprine) or cytotoxic agents such as cytotoxan or methotrexate.

[0035] Activated partial thromboplastin time (APTT) Assay: An assay thatmeasures the time it takes plasma to clot. APTT is the period requiredfor clot formation in recalcified blood plasma after contact activationand the addition of platelet substitutes (e.g., brain cephalin orsimilar phospholipids). In normal individuals, the APTT is about 30-40seconds, and the PTT (therapeutic level) is 60 to 70 seconds. Aprolonged APTT can indicate a deficiency of a number of clotting factorsincluding Factors XII, XI, IX, VIII, X, V, and II, and fibrinogen.

[0036] Bleeding Time Assay: An assay used to measure the amount of timeit takes for a subject's blood to clot. A blood pressure cuff is placedon the upper arm and inflated. Two incisions are made on the lower arm.These are about 10 mm (less than ½ inch) long and 1 mm deep (just deepenough to cause minimal bleeding). The blood pressure cuff isimmediately deflated. Blotting paper is touched to the cuts every 30seconds until the bleeding stops. The length of time it takes for thecuts to stop bleeding is recorded.

[0037] In normal, non-hemophiliacs, bleeding stops within about one toten minutes and may vary from lab to lab, depending on how the assay ismeasured. In contrast, severe hemophiliacs having less than 1% of normallevels of the appropriate clotting factor have a whole blood clottingtime of greater than 60 minutes.

[0038] In mice, the bleeding time is assayed by transecting the tip ofthe tail and periodically touching a blotting paper until a clot isformed at the tip of the tail. Normal bleeding time is between 2-4minutes. In contrast, hemophiliac mice having less than 1% of normallevels of the appropriate clotting factor have a bleeding time ofgreater than 15 minutes (see FIGS. 7A, 7B, 8A and 8B).

[0039] cDNA (complementary DNA): A piece of DNA lacking internal,non-coding segments (introns) and regulatory sequences which determinetranscription. cDNA can be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

[0040] Chemical synthesis: An artificial means by which one can make aprotein or peptide. A synthetic protein or peptide is one made by suchartificial means.

[0041] Clotting disorder: A disease resulting in a defect in hemostasisin a subject. In one embodiment, it is a disease of the blood clotting(coagulation) system in which bleeding is prolonged due to inadequateclotting factors in the blood, for example inadequate expression of theclotting factor, such as inadequate expression due to a geneticabnormality. Normal blood hemostasis is a complex process involving asmany as 20 different plasma proteins, known as clotting factors.Normally, a complex chemical process occurs using these clotting factorsto form a substance called fibrin that stops bleeding. When certaincoagulation factors are deficient or missing, the process does not occurnormally. Bleeding problems can range from mild to severe.

[0042] Some bleeding disorders are present at birth and in someinstances are inherited disorders. Specific examples include, but arenot limited to: hemophilia A, hemophilia B, protein C deficiency, andVon Willebrand's disease. Some bleeding disorders are developed duringcertain illnesses (such as vitamin K deficiency, severe liver disease),or treatments (such as use of anticoagulant drugs or prolonged use ofantibiotics).

[0043] Clotting factor: Includes any protein which promotes properhemostasis. In one embodiment, a clotting factor is factor VIII (F.VIII)or factor IX (F.IX), or a variant or fragment thereof which retains itshemostatic activity, for example as measured using an APTT assay or ableeding time assay. In another embodiment, when orally administered ina therapeutically effective amount, the clotting factor increaseshemostasis in a subject suffering from a clotting disorder, such ashemophilia. In a particular embodiment, a clotting factor is arecombinant clotting factor, wherein expression of a DNA encoding theclotting factor in a mammal results in the presence of the recombinantclotting factor protein in the milk of the transgenic mammal.

[0044] Comprises: A term that means “including.” For example,“comprising A or B” means including A or B, or both A and B, unlessclearly indicated otherwise.

[0045] Deletion: The removal of a sequence of a nucleic acid, forexample DNA, the regions on either side being joined together.

[0046] DNA: Deoxyribonucleic acid. DNA is a long chain polymer whichcomprises the genetic material of most living organisms (some viruseshave genes comprising ribonucleic acid, RNA). The repeating units in DNApolymers are four different nucleotides, each of which comprises one ofthe four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides, referred to as codons, in DNA molecules code for amino acidin a polypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

[0047] Enhance: To improve the quality, amount, or strength ofsomething. In one embodiment, a therapy enhances or increases hemostasisin a subject (such as a hemophiliac) if the subject is more effective atblood clotting (for example, their blood clotting time decreases). Suchenhancement can be measured using the methods disclosed herein, forexample determining the bleeding time of a subject using an APTT orbleeding time assay.

[0048] Factor VIII (F.VIII) F.VIII is a protein required for theefficient clotting of blood, and functions in coagulation as a cofactorin the activation of factor X by factor IX. A concentration of about 100ng/ml for F.VIII in the blood is considered in the normal range.Deficiency of F.VIII is associated with hemophilia A, and severe formsof the disease can result when a subject has less than about 1% of thenormal amount of F.VIII (i.e. less than about 1 ng of F.VIII per ml ofblood). F.VIII is synthesized as a 2351 amino acid single chainprecursor protein, which is proteolytically processed. The human factorVIII gene (186,000 base-pairs) consists of 26 exons ranging in size from69 to 3,106 bp and introns as large as 32.4 kilobases (kb). Examples ofF.VIII nucleic acid and protein sequences, including variants andfragments thereof, and sequences from different organisms, are publiclyavailable on Genbank (for example see Accession Nos: K01740, M14113, andE00527 (human); AF016234 (canine); and L05573 (mouse)).

[0049] Gaucher Disease: An autosomal-recessive disorder that resultsfrom defective activity of acid β-glucosidase. Disease variants areclassified based on the absence or presence and severity ofneuronopathic involvement. Enzyme therapy is currently the treatment ofchoice in significantly affected patients. For example, cerezyme, arecombinantly produced mannose-terminated (macrophage-targeted) acidβ-glucosidase, is currently used to diminish hepatosplenomegaly andimprove bone marrow involvement and hematologic findings. However, it ispossible that oral administration of β-glucosidase using the methodsdisclosed herein, can be used to alleviate one or more symptomsassociated with Gaucher disease described below.

[0050] Type 2 Gaucher disease is a rare, severe CNS disease that leadsto death by 2 years of age.

[0051] Type 3 Gaucher disease has highly variable manifestations in theCNS and viscera. It can present in early childhood with rapidlyprogressive, massive visceral disease and slowly progressive to staticCNS involvement; in adolescence with dementia; or in early adulthoodwith rapidly progressive, uncontrollable myoclonic seizures and mildvisceral disease. Visceral disease in type 3 is nearly identical to thatin type 1, but is generally more severe. Early CNS findings may belimited to defects in lateral gaze tracking, which may remain static fordecades. Mental retardation can be slowly progressive or static.

[0052] Type 1 Gaucher disease is a highly variable nonneuronopathicdisease. Younger patients tend to have a greater degree ofhepatosplenomegaly and accompanying blood cytopenias. Older patientshave a greater tendency for chronic bone disease. Hepatosplenomegalyoccurs in virtually all symptomatic patients and can be minor ormassive. Accompanying anemia and thrombocytopenia are variable and notlinearly related to liver or spleen volume. Severe liver dysfunction isunusual, though minor liver function abnormalities are common. Splenicinfarctions can resemble an acute abdomen. Pulmonary hypertension andalveolar Gaucher cell accumulation are uncommon, but life-threatening.

[0053] Though it is more common in adult patients, clinically evidentskeletal disease in children can be devastating, resulting in massivedestruction of the axial and peripheral skeleton. All patients withGaucher disease have non-uniform infiltration of bone marrow bylipid-laden macrophages, termed Gaucher cells. This can lead to marrowpacking with subsequent infarction, ischemia, necrosis, and corticalbone destruction. Bone marrow involvement spreads from proximal todistal in the limbs and can involve the axial skeleton extensively,causing vertebral collapse. In addition to bone marrow involvement, boneremodeling is defective, with loss of total bone calcium leading toosteopenia, osteonecrosis, avascular infarction, and vertebralcompression fractures and spinal cord involvement. Aseptic necrosis ofthe femoral head is common, as is fracture of the femoral neck.

[0054] Affected patients experience chronic, ill-defined bone pain thatcan be debilitating. Some patients have one or more “bone crises” intheir lifetimes that are associated with localized, excruciating pain,and, on occasion, local erythema, fever, and leukocytosis. Any bone canbe involved, though the femurs and vertebral bodies are affected mostoften. These crises represent acute infarctions of bone, as evidenced innuclear scans by localized absent uptake of pyrophosphate agents. X-raysare usually negative initially but may show lytic lesions 4 to 6 monthsafter the acute phase.

[0055] The diagnosis of Gaucher disease is established by demonstratingdecreased acid β-glucosidase activity (0 to 20% of normal) in nucleatedcells. The enzyme is not present in bodily fluids. The sensitivity ofenzyme testing is poor for detecting heterozygous carriers; moleculartesting is preferred when the mutations are known. Four common mutationsaccount for ˜90 to 95% of the mutations in affected patients: N370S(1226G), 84GG (a G insertion at cDNA position 84), L444P (1448C), andIVS-2 (an intron 2 splice junction mutation). Genotype/phenotype studiesindicate a significant correlation, though not absolute, between diseasetype and severity and the acid β-glucosidase genotype.

[0056] Factor IX (F.IX): F.IX is a vitamin K-dependent protein requiredfor the efficient clotting of blood, and functions in coagulation as anactivator of factor X. A concentration of about 1-5 μg/ml of F.IX in theblood is considered in the normal range. Deficiency of F.IX isassociated with hemophilia B, and severe cases result when theconcentration of F.IX is less than about 1% of the normal concentrationof F.IX (i.e. less than about 0.01-0.05 μg F.IX per ml of blood). CanineF.IX possesses 86% identity at the amino-acid level with human F.IX(Evans et al. 1989. Blood 74:207-12). F.IX nucleic acid and proteinsequences, including variants and fragments thereof, and sequences fromdifferent organisms, are publicly available (for example see Kurachi etal., 1982. Proc. Nat. Acad. Sci. U.S.A. 79(21):6461-4; Genbank AccessionNos: J00136, XM_(—)045316, K02402, J00137, and M11309 (human) and M21757and M33826 (canine)).

[0057] Hemophilia: A blood coagulation disorder caused by a deficientclotting factor activity, which decreases hemostasis. Severe formsresult when the concentration of clotting factor is less than about 1%of the normal concentration of the clotting factor in a normal subject.In some subjects, hemophilia is due to a genetic mutation which resultsin impaired expression of a clotting factor. In others, hemophilia is anauto-immune disorder, referred to as acquired hemophilia, in which theantibodies which are generated against a clotting factor in a subjectresult in decreased hemostasis.

[0058] Hemophilia A results from a deficiency of functional clottingfactor VIII (F.VIII), while hemophilia B results from a deficiency offunctional clotting factor IX (F.IX). These conditions which are due toa genetic mutation are caused by an inherited sex-linked recessive traitwith the defective gene located on the X chromosome, and this disease istherefore generally found only in males. The severity of symptoms canvary with this disease, and the severe forms become apparent early on.Bleeding is the hallmark of the disease and typically occurs when a maleinfant is circumcised. Additional bleeding manifestations make theirappearance when the infant becomes mobile. Mild cases may go unnoticeduntil later in life when they occur in response to surgery or trauma.Internal bleeding may happen anywhere, and bleeding into joints iscommon.

[0059] Hemophiliac: A subject having a hemophilia.

[0060] Hemostasis: Arrest of bleeding blood by blood clot formation.When hemostasis is increased, it is increased relative to hemostasis ina subject prior to orally administering a therapeutically effectiveamount of the appropriate clotting factor to the subject. In oneembodiment, when hemostasis increases in a subject with hemophilia, theblood clotting time decreases. Blood clotting time is the length of timeit takes for peripheral blood to clot using an activated partialthromboplastin time assay (APTT, see EXAMPLE 9) or by measuring bleedingtime (see above and EXAMPLE 5). In a particular embodiment, the bloodclotting time decreases by at least 50%, for example at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99% or even about 100% (i.e. the blood clotting timeis similar to what is observed for a normal subject) when compared tothe blood clotting time of the subject prior to orally administering tothe subject a therapeutically effective amount of the appropriateclotting factor. In yet another embodiment, the blood clotting time inthe affected subject is corrected to about 90% of a normal subject, forexample to about 95%, for example about 100%, after oral administrationof a therapeutically effective amount of the appropriate clottingfactor.

[0061] Impaired Expression: Decreased expression of a DNA or proteinresulting in a deficiency of the protein in a subject. Such a proteindeficiency may cause disease in the subject.

[0062] Isolated: An isolated nucleic acid has been substantiallyseparated or purified away from other nucleic acid sequences in the cellof the organism in which the nucleic acid naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA. The term “isolated” thusencompasses nucleic acids purified by standard nucleic acid purificationmethods. The term also embraces nucleic acids prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

[0063] Liposome: A closed, solvent-filled vesicle bounded by a singlebilayer of phospholipids, which is impermeable to many substances. Inone embodiment, a liposome has the properties described in U.S. Pat. No.4,348,384 to Horikoshi et al. (herein incorporated by reference).

[0064] Mammal: This term includes both human and non-human mammals.Similarly, the terms “patient,” “subject,” and “individual” includesboth human and veterinary subjects. Examples of mammals include, but arenot limited to: humans, pigs, cows, goats, cats, dogs, rabbits and mice.

[0065] Normal Cells: Non-disease cells. In one embodiment, normal cellsare cells obtained from a healthy subject, for example as compared to ahemophilic or diabetic subject.

[0066] Normal Subject: A subject who does not have an antigen-deficiencydisease. For example, a subject who does not have hemophilia A or B.

[0067] Oligonucleotide: A linear polynucleotide sequence of up to about200 nucleotide bases in length, for example a polynucleotide (such asDNA or RNA) which is at least about 6 nucleotides, for example at least15, 50, 100 or 200 nucleotides long.

[0068] Operably linked: A first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

[0069] ORF (open reading frame): A series of nucleotide triplets(codons) coding for amino acids without any termination codons. Thesesequences are usually translatable into a peptide.

[0070] Oral administration: A method of delivering an agent to a subjectby mouth. In one embodiment, oral administration is achieved by feedingthe agent to the subject. In a particular embodiment, the agent is anantigen, such as a protein, for example F.VIII or F.IX. The agent can bedelivered with one or more pharmaceutically acceptable carriers whichare not liposomes. In addition, the agent and one or moretherapeutically effective pharmaceutical compounds can be administeredconcurrently or separately.

[0071] Oral tolerance: A method of downregulating an immune response ina subject by orally administering an antigen (i.e. by feeding) to thesubject. Oral tolerance is characterized by decreased levels of systemicantibody production, as well as decreased delayed type hypersensitivityresponses (DTH), T cell proliferation, cytotoxic responses and graftrejection (Alpan et al. 2001. J. Immunol. 166:4843-52; Chen et al. 1995.Nature 376:177-80; Weiner. 1997. Imm. Today. 7:335-44; Sayegh et al.1992. Transplantation. 53:163-6).

[0072] The tolerogenic effect due to administration of high doses oralantigen is deletion of antigen-specific T cells by apoptosis (Weiner.1997. Imm. Today. 7:335-44). Administration of low doses of oral antigeninduces a switch in effector class such that Th1 type responses (DTH,cytotoxicity, graft rejection and complement fixing antibodies) aresupplanted by Th3 type responses (the production of IL-4, IL10, TGF-βand, occasionally, secretory IgA) (Weiner. 1997. Imm. Today. 7:335-44).Low dose oral tolerance reduces the severity of EAE (Fields et al. 2000.Molecular Therapy. 3:225-35) and enables the acceptance of foreigntransplants.

[0073] Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription.

[0074] Purified: The term purified does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedclotting factor preparation is one in which the factor is more pure thanthe factor in its natural environment within a cell. For example, apreparation of a clotting factor protein is purified if the proteinrepresents at least 50%, for example at least 70%, of the total proteincontent of the preparation. Methods for purification of proteins andnucleic acids are well known in the art. Examples of methods that can beused to purify an antigen, such as a clotting factor include, but arenot limited to the methods disclosed in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 17);U.S. Pat. No. 6,005,082 to Smeds; EP 0294910 to van Ooyen et al.;

[0075] Recombinant: A recombinant nucleic acid is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques. Arecombinant protein is one that results from expressing a recombinantnucleic acid encoding the protein.

[0076] RT: Room temperature

[0077] Sample: Biological samples containing genomic DNA, cDNA, RNA, orprotein obtained from the cells of a subject, such as those present inperipheral blood, urine, saliva, tissue biopsy, surgical specimen, fineneedle aspriates, amniocentesis samples and autopsy material.

[0078] Sequence identity/similarity: The identity/similarity between twoor more nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are.

[0079] Methods of alignment of sequences for comparison are well knownin the art. Various programs and alignment algorithms are described in:Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J.Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids. Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

[0080] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

[0081] For comparisons of amino acid sequences of greater than about 30amino acids, the Blast 2 sequences function is employed using thedefault BLOSUM62 matrix set to default parameters, (gap existence costof 11, and a per residue gap cost of 1). When aligning short peptides(fewer than around 30 amino acids), the alignment should be performedusing the Blast 2 sequences function, employing the PAM30 matrix set todefault parameters (open gap 9, extension gap 1 penalties). Proteinswith even greater similarity to the reference sequence will showincreasing percentage identities when assessed by this method, such asat least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity.When less than the entire sequence is being compared for sequenceidentity, homologs will typically possess at least 75% sequence identityover short windows of 10-20 amino acids, and can possess sequenceidentities of at least 85%, 90%, 95% or 98% depending on their identityto the reference sequence. Methods for determining sequence identityover such short windows are described at the NCBI web site.

[0082] Protein homologs are typically characterized by possession of atleast 70%, such as at least 75%, 80%, 85%, 90%, 95% or even 98% sequenceidentity, counted over the full-length alignment with the amino acidsequence using the NCBI Basic Blast 2.0, gapped blastp with databasessuch as the nr or swissprot database. Queries searched with the blastnprogram are filtered with DUST (Hancock and Armstrong, 1994, Comput.Appl. Biosci. 10:67-70). Other programs use SEG.

[0083] One of skill in the art will appreciate that these sequenceidentity ranges are provided for guidance only; it is possible thatstrongly significant homologs could be obtained that fall outside theranges provided. Provided herein are the peptide homologs describedabove, as well as nucleic acid molecules that encode such homologs.

[0084] Nucleic acid sequences that do not show a high degree of identitymay nevertheless encode identical or similar (conserved) amino acidsequences, due to the degeneracy of the genetic code. Changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein. Such homologous peptides can, for example, possess at least75%, 80%, 90%, 95%, 98%, or 99% sequence identity determined by thismethod. When less than the entire sequence is being compared forsequence identity, homologs can, for example, possess at least 75%, 85%90%, 95%, 98% or 99% sequence identity over short windows of 10-20 aminoacids. Methods for determining sequence identity over such short windowscan be found at the NCBI web site. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is possible that significant homologs or other variants can beobtained that fall outside the ranges provided.

[0085] Subject: Living multicellular vertebrate organisms, a categorywhich includes, both human and veterinary subjects for example, mammals,rodents, and birds.

[0086] Therapeutically Effective Amount: An amount sufficient to achievea desired biological effect, for example an amount that is effective toincrease hemostasis. In particular examples, it is a concentration ofclotting factor, such as F.VIII or F.IX, effective to increasehemostasis, such as in a subject to whom it is administered, such as ahemophiliac. In other examples, it is an amount effective to increasehemostasis by more than a desired amount.

[0087] In one embodiment, the therapeutically effective amount alsoincludes a quantity of clotting factor (such as a clotting factorprotein or nucleic acid) sufficient to achieve a desired effect in asubject being treated. For instance, these can be an amount necessary toimprove signs and/or symptoms a disease such as hemophilia, for exampleby increasing hemostasis.

[0088] An effective amount of a clotting factor can be administered in asingle dose, or in several doses, for example daily, during a course oftreatment. However, the effective amount of clotting factor will bedependent on the source of clotting factor administered (i.e. clottingfactor isolated from a blood sample versus recombinant clotting factorexpressed in milk), the subject being treated, the severity and type ofthe condition being treated, and the manner of administration clottingfactor. For example, a therapeutically effective amount of clottingfactor can vary from about 0.01 mg/kg body weight to about 5 g/kg bodyweight, for example, at least 5 mg/kg daily, for example at least 50mg/kg daily. In other embodiment, it is a concentration of F.VIII, whenorally administered, which results in a blood concentration of greaterthan about 1 ng F.VIII per ml blood, for example greater than about 10ng per ml, for example about 100 ng per ml. In yet another embodiment,it is a concentration of F.IX, when orally administered, which resultsin a blood concentration of greater than about 0.01-0.05 μg F.IX per mlblood, for example greater than about 0.1-0.5 μg per ml, for exampleabout 1-5 μg per ml.

[0089] The methods disclosed herein have equal application in medicaland veterinary settings. Therefore, the general term “subject beingtreated” is understood to include all animals (e.g. humans, apes, dogs,cats, horses, and cows) that require an increase in the desiredbiological effect, such as enhanced hemostasis susceptible to clottingfactor-mediated modulation.

[0090] Therapeutically effective dose: A dose of antigen sufficient toincrease the amount of that antigen in a subject to whom it isadministered, resulting in a regression of a pathological condition, orwhich is capable of relieving signs or symptoms caused by the condition.In a particular embodiment, it is a dose of clotting factor sufficientto increase hemostasis in a hemophiliac.

[0091] Transformed: A transformed cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Asused herein, the term transformation encompasses all techniques by whicha nucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

[0092] Transgenic Cell: Transformed cells which contain foreign,non-native DNA.

[0093] Transgenic mammal: Transformed mammals which contain foreign,non-native DNA. In one embodiment, the non-native DNA is an antigen,such as human F.VIII or human F.IX. In a particular embodiment, atransgenic mammal expresses a recombinant clotting factor in its milk.In yet another embodiment, the transgenic animal is a pig expressingrecombinant human F.VIII or F.IX in its milk. One skilled in the artwill understand that any mammal can be used, including, but not limitedto pigs, cows, goats, sheep, rats, mice, rabbits, dogs, cats, andprimates.

[0094] Variants or fragments or fusion proteins: The production ofprotein which is orally administered can be accomplished in a variety ofways (for example see EXAMPLES 15 and 16). DNA sequences which encodefor a protein or fusion protein, or a fragment or variant of a protein(for example a fragment or variant having 80%, 90% or 95% sequenceidentity to a blood clotting factor) can be engineered to allow theprotein to be expressed in eukaryotic cells or organisms, bacteria,insects, and/or plants. To obtain expression, the DNA sequence can bealtered and operably linked to other regulatory sequences. The finalproduct, which contains the regulatory sequences and the therapeuticprotein, is referred to as a vector. This vector can be introduced intoeukaryotic, bacteria, insect, and/or plant cells. Once inside the cellthe vector allows the protein to be produced.

[0095] A fusion antigen comprising a protein, such as F.VIII or F.IX (orvariants, polymorphisms, mutants, or fragments thereof) linked to otheramino acid sequences that do not inhibit the desired activity of theprotein, for example the ability to increase hemostasis. In oneembodiment, the other amino acid sequences are no more than 10, 20, 30,or 50 amino acid residues in length.

[0096] One of ordinary skill in the art will appreciate that the DNA canbe altered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes an antigen. Such variants can bevariants optimized for codon preference in a host cell used to expressthe protein, or other sequence changes that facilitate expression.

[0097] Vector: A nucleic acid molecule as introduced into a host cell,thereby producing a transformed host cell. A vector can include nucleicacid sequences that permit it to replicate in the host cell, such as anorigin of replication. A vector can also include one or more selectablemarker genes and other genetic elements known in the art.

[0098] Additional definitions of terms commonly used in moleculargenetics can be found in Benjamin Lewin, Genes V published by OxfordUniversity Press, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyaid Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

[0099] Disclosed herein is a method for increasing hemostasis in asubject having a hemophilia by orally administering to the subject atherapeutically effective amount of a clotting factor not present in aliposome, sufficient to induce oral tolerance and supply exogenousclotting factor to the subject. In one embodiment, hemophilia is aresult of impaired expression of a clotting factor. In yet anotherembodiment, increasing hemostasis comprises decreasing blood clottingtime. For example, blood clotting time can be decreased by at least 50%,for example at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or even at least 99% compared toa blood clotting time of an untreated hemophilic subject. The bloodclotting time can be measured using an activated partial thromboplastintime (APTT) assay or by a bleeding time assay.

[0100] In another embodiment, the clotting factor is administered inabsence of an anti-immune therapy. In a particular embodiment, atherapeutically effective amount of a clotting factor is a daily dose ofat least 5 mg of clotting factor per kg of subject, for example at least50 mg of clotting factor per kg of subject. The clotting factor can beadministered daily or every other day. The subject to be treated can bea mammal, for example a human or dog. The hemophilia can be hemophilia Aand the clotting factor F.VIII, or hemophilia B and the clotting factorF.IX. In one embodiment, a therapeutically effective amount of F.VIII isat least 5 mg of clotting factor per 1 kg of subject daily and atherapeutically effective amount of F.IX is at least 50 mg of clottingfactor per 1 kg of subject daily.

[0101] In a particular embodiment, oral administration is achieved byfeeding the subject a therapeutically effective amount of the clottingfactor. For example, the clotting factor can be a recombinant clottingfactor present in milk of a transgenic mammal, which is fed to ahemophiliac. In one embodiment, the clotting factor, such as F.IX, ispresent in milk obtained from a transgenic pig expressing the clottingfactor, such as recombinant F.IX. The clotting factors can be present inat least one pharmaceutically effective carrier, such as water or milk.

[0102] The methods disclosed herein can further include administering toa subject an isolated recombinant nucleic acid molecule encoding aclotting factor, such that the clotting factor is expressed in thesubject.

[0103] Also disclosed herein is a method for increasing hemostasis in asubject having a hemophilia, which includes, and in some examplesconsists of, orally administering to the subject a therapeuticallyeffective amount of a clotting factor and at least one pharmaceuticallyacceptable carrier which is not a liposome. In other examples, thepharmaceutical compositions disclosed herein (including clottingfactors) can be administered in combination, or separately, with one ormore other therapeutic treatments, such as other agents that increasehemostasis.

[0104] A method is also disclosed for orally administering a recombinantclotting factor to a subject for treatment of a hemophilia by orallyadministering milk containing recombinant clotting factor to the subjectat a therapeutically effective amount such that hemostasis in thesubject is increased, thereby treating the hemophilia. In oneembodiment, the milk contains at least 2 g recombinant clottingfactor/liter of milk. At this concentration, a 70 kg human would drinkabout 0.175 liters of milk daily to achieve an effective dose of aboutat least 5 mg/kg or about 1.75 liters of milk to achieve an effectivedose of about at least 50 mg/kg. The milk can be obtained from atransgenic mammal, such as a pig which expresses the clotting factor (orother therapeutic protein) in its milk. In a particular example, themilk can contain recombinant F.IX antigen at a concentration of about 2g per liter, for example about 2.5 g per L, either expressed in the milkor exogenously added to the milk.

[0105] Also disclosed herein is a method for orally administering arecombinant clotting factor to a subject for treatment of a hemophiliaby expressing the clotting factor in a mammal such that the clottingfactor is expressed in milk of the mammal and orally administering themilk in which the clotting factor has been expressed to the subject at atherapeutically effective amount such that blood clotting time in thesubject is reduced, thereby treating the hemophilia. In a particularembodiment, the hemophilia is hemophilia B and the clotting factor isF.IX.

[0106] Further disclosed is a method for orally administering arecombinant F.IX protein to a subject for treatment of hemophilia B byorally administering milk containing recombinant F.IX protein at atherapeutically effective amount such that blood clotting time in thesubject is reduced, thereby treating the hemophilia B.

[0107] Also disclosed is a method of orally administering to ahemophiliac a therapeutically effective amount of a clotting factorprotein sufficient to induce oral tolerance to the clotting factorprotein and supply exogenous clotting factor protein to the hemophiliac,wherein the clotting factor protein is not present in a liposome, andwherein oral tolerance prepares the hemophiliac for in vivo expressionof a clotting factor gene. In a particular embodiment, the clottingfactor is F.VIII or F.IX.

[0108] Disclosure of certain specific examples is not meant to excludeother embodiments.

EXAMPLE 1 Ovalbumin Oral Tolerance

[0109] This example describes the methods used to induce oral tolerancein mice to ovalbumin (OVA) by first orally administering OVA prior toimmunization with OVA. Similar methods can be used to test any antigenof interest, such as F.VIII, F.IX, protein C, or insulin, in anyanimal/subject of interest.

[0110] Normal B10 mice were fed OVA by adding the protein to theirdrinking water for eight days at concentrations of 0.02, 0.2 or 2 mg/ml.The mice drank an average of 5.2 ml/mouse/day. Therefore, the doses were0.1, 1.0 and 10 mg/mouse/day of OVA. Control mice only drank water. Twodays after the last feeding, all mice were immunized with 50 μg OVA incomplete Freund's adjuvant (CFA) (Sigma, St. Louis, Mo.) in the leftfootpad. Seven days later, footpad swelling was determined by measuringthe thickness of the footpad. Measurement of the right (un-injected)footpad served as an untreated control. The footpad swelling responseswere compared between injected, OVA-fed mice and injected, non-OVA fedcontrol mice. As shown in FIG. 1A, the footpads of OVA-fed mice swelledless than those of the unfed group.

[0111] To compare the amount of IL4, TGF-β, and IFN-γ production, micewere fed 0.1 mg OVA for 10 days, then immunized as described above.Seven days after immunization, lymph nodes draining the immunizationsite were harvested and cultured in vitro with 100 μg/ml OVA. Theculture was carried in 96 well round bottom plates, with 500,000 cellsper well, for four days. For the last 8 hours of the culture, cells werepulsed with tritiated thymidine to assay for proliferation using a betaplate cell harvester. For IL-4 and IFN-γ, supernatants were assayed atday four. For TGF-β, after four days in culture, cells were washed,restimulated with APC and OVA in serum-free medium, and supernatantsassayed at day three. ELISA assays were performed using commerciallyavailable kits from Endogen (Rockford, Ill.), according to themanufacturer's instructions. As shown in FIG. 1B, OVA-fed mice made highlevels of IL-4 and TGF-β, but low levels of IFN-γ compared to mice inthe unfed group.

[0112] To compare the amount of T-cell proliferation, mice were feddoses of OVA ranging from 0.001-10 mg every-other day for five feeds andimmunized with 50 μg OVA in CFA. Seven days after immunization, lymphnodes draining the immunization site were harvested by surgicaldissection. CD4 T-cells were purified from the lymph nodes and culturedin vitro with irradiated spleen cells and a graded concentration of OVAas described above. As shown in FIG. 1C, T cells in the draining lymphnodes of OVA-fed mice proliferated less than those mice in the unfedgroup. Since T cells in lymph nodes proliferate in response to regionalantigenic stimulation, the reduction in T cell proliferation was ameasure of decreased antigenic stimulation from OVA injection in thefootpad.

EXAMPLE 2 Factor IX Oral Tolerance

[0113] This example describes experiments in which mice developed oraltolerance to F.IX by orally administering F.IX prior to immunizationwith F.IX, using the methods described in EXAMPLE 1.

[0114] Briefly, normal B10.A mice (F.IX−/+) or hemophilia B mice(F.IX−/−) were fed transgenic human F.IX (hF.IX) in pig milk (seeEXAMPLE 5) or control pig milk (no hF.IX) every other day for threeweeks. Individual mice drank about 4 ml (10 μg hF.IX/day/mouse) of themilk at each time. Two days after the last feeding, all mice wereimmunized with 50 μg human F.IX emulsified in CFA at the base of thetail. Eight days later, the amount of T cell proliferation wasdetermined by culturing T-cells from the lymph nodes with gradedconcentrations of human F.IX (purified from the transgenic pig milk) forfour days using the methods described in EXAMPLE 1. As shown in FIGS. 2Aand 2B, feeding mice human F.IX had a similar effect to the resultobserved in EXAMPLE 1 for OVA, in that it decreased T cell proliferationupon subsequent immunization with human F.IX in CFA.

[0115] To demonstrate that oral administration of F.IX induces localimmunity, hemophiliac B mice were fed transgenic F.IX in milk (seeEXAMPLE 5) while normal mice did not receive F.IX, as described above.Subsequently the level of intestinal IgA to F.IX was measured bycollecting stool samples, dissolving them in PBS, and assaying for IgAusing standard ELISA methods. As shown in FIG. 2C, feeding F.IX induceslocal immunity as measured by the production of secretory IgA.

EXAMPLE 3 Antibodies Produced by Orally Tolerized Hemophilia B Mice DoNot Result in Clinical Resistance to Infused F.IX

[0116] To measure IgG1 and IgG2a antibodies specific for hF.IX, plateswere coated with hF.IX (10 μg/ml in PBS) then blocked with BSAovernight. After five washes, serum samples from the normal andhemophilia B mice fed as described in EXAMPLE 2 were added and incubatedfor two hours. The plates were then washed and subsequently incubatedwith HRP-conjugated goat-anti-mouse IgG1 and IgG2a for two hours,followed by TMP for color development, and plates subsequently read onan ELISA plate reader. As shown in FIGS. 3A and 3B, whereas unfedcontrol mice made strong IgG2a antibodies, mice fed with hF.IX made nodetectable IgG2a, but did produce IgG1.

[0117] Therefore, in normal mice, oral administration of human F.IX didnot completely inhibit the production of small amounts of antibodiesafter immunization in CFA, as detected in ELISA assays, although itcaused a shift from IgG2a to IgG1 (FIGS. 3A and 3B). However, CFA is thestrongest adjuvant known, and may elicit antibody production that wouldnot occur under conditions that more closely resemble a clinicalsituation. However, antibodies detectable in ELISA assays may bind todenatured proteins, but may not act as inhibitors of the native protein.Therefore, whether orally tolerized hemophiliac mice would madeantibodies after CFA immunization, and whether these antibodies wouldact as inhibitors and lead to resistance to treatment, was determined.

[0118] Hemophilia B mice were fed transgenic F.IX milk, or fed milkwithout F.IX, every other day for two weeks. Two days after the lastfeeding, mice were immunized with h.FIX in CFA as described above. Ninedays later, the tail vein was infused with a dose of F.IX just over theborderline for correcting the bleeding time (0.13 IU of functional hF.IXpurified from the pig milk). This amount was chosen because it is in therange of F.IX most sensitive to inhibitors. Bleed times were measuredsix hours later. As shown in FIG. 4, group 2 shows that this dose ofhF.IX partially corrected the bleeding time (clotting occurred within7-10 minutes). This clotting time is about twice as long as the 4-6minutes clotting time of normal mice (see FIG. 7A), showing that 0.13 IUof functional hF.IX is a highly sensitive range of factor. Bleedingtimes in the immunized unfed F.IX hemophilia B mice (FIG. 4, group 3)were as long as those in mice not given factor (FIG. 4, group 1),showing that the antibodies elicited by a single immunization with hF.IXin CFA were highly inhibitory. However, the F.IX knock-out mice(hemophilia B mice) that were orally tolerized before immunization (FIG.4, group 4) showed little evidence of inhibitors, as their bleedingtimes were only slightly longer than those of infused mice that had notbeen immunized.

[0119] These results demonstrate that although orally tolerized miceproduce antibodies when immunized with a very strong adjuvant, theseantibodies do not lead to clinical resistance to hF.IX infusion.Therefore, the antibodies may be specific for denatured forms of thefactor that occur in the adjuvant mixture and on ELISA plates, but theantibodies are not inhibitory to the native form of hF.IX in vivo.

EXAMPLE 4 Casein Oral Tolerance

[0120] This example describes the methods used to determine if sheepwould develop oral tolerance to casein using the methods described inEXAMPLES 1 and 2. Similar methods can be used to test any antigen ofinterest, such as F.VIII, F.IX, protein C, or insulin, in anyanimal/subject of interest.

[0121] Lambs were raised on sheep or cow milk. After 3-4 months, lambswere immunized with 0.5 mg sheep casein in CFA. Three and six weekslater, lambs were boosted with 0.5 mg casein in incomplete Freund'sadjuvant (IFA) (Sigma). Two months after the last immunization,peripheral blood mononuclear cells (PBMCs) were assayed forproliferation in the presence of titrated doses of sheep casein. SheepPBMCs were isolated by centrifugating the blood through a lymphoprepgradient. Cells layered on the lymphoprep were collected and cultured invitro in 96 well plates for four days in the presence of casein.Proliferation was measured by pulsing the cells with tritiated thymidineduring the last eight hours of the culture and reading the results on abeta plate counter.

[0122] To measure casein-specific IgG in serum, maleic anhydrideactivated polystyrene plates were coated with sheep casein (10 μg/ml)for 30 minutes at 37° C. and blocked with PBS 1% BSA (bovine serumalbumin) for 30 minutes. After discarding the blocking buffer and fivewashes, serum samples ({fraction (1/10)} diluted) were added into thewells and incubated for two hours. The plates were subsequently washed,and horseradish peroxidase (HRP) conjugated rabbit anti-sheep IgG (cloneN126E, Southern Biotechnology Associates) was added and incubated fortwo hours, followed by TMP for color development, and platessubsequently read on an ELISA plate reader.

[0123] As shown in FIGS. 5A-5C, oral tolerance occurred in sheep. PBMCsfrom lambs that drank their mother's milk (sheep milk) did notproliferate in vitro to a sheep milk protein, casein, upon immunizationas young adults (FIG. 5A). In contrast, lambs that were fostered oncow's milk showed a vigorous proliferative response (FIG. 5B). Inaddition, orally tolerized lambs failed to make antibodies to casein(FIG. 5C).

EXAMPLE 5 Orally Administered Clotting Factors Remain Functional

[0124] Having demonstrated that oral tolerance to several differentantigens can be achieved, it was then demonstrated that the antigenremained functional after passing through the digestive system into thebloodstream. This example describes methods that were used to test thefunctionality of F.VIII and F.IX after they were orally administered.Similar methods can be used to identify a therapeutically effectiveamount of clotting factor necessary to increase hemostasis in a subject,such as in a hemophiliac.

[0125] Factor IX

[0126] Normal B10.A mice were fed either 0.5 ml of human F.IX intransgenic pig milk (which contained about 2.5 g/L of human F.IX; seeVan Cott et al. 1999. Genet. Anal. 15:155-60) or water alone. One hourafter feeding, plasma was obtained from the mice and added to sheep Tcells that are reactive against human F.IX, to determine if the plasmacould stimulate the sheep PBMCs. Sheep T cells reactive against humanF.IX were generated by immunizing sheep with human F.IX (100 μg) in CFA.PBMC from these sheep proliferate in a dose-dependent manner to purifiedhuman F.IX in vitro (FIG. 4A, dotted line) and therefore can be used tomeasure the amounts of human F.IX in blood samples. The plasma wasdiluted from about 1:10 and decrease in 10-fold dilutions down to 1:10⁷.Cells were cultured for four days in 96 well plates and pulsed withtritiated thymidine the last eight hours of the culture. As a control,sheep T cells were cultured in the presence of purified human F.IX.

[0127] As shown in FIG. 6A, F.IX present in the pig milk passed into theplasma after feeding. Sheep PBMC responded strongly to the fed mouseplasma (FIG. 6A, solid line), but not to plasma from unfed mice (FIG.6A, bottom dashed line). It was determined that the fed mouse plasmacontained about 6.7 μg/ml of human F.IX, indicating that about 0.1% ofthe fed F.IX entered the circulation. This result was confirmed by asandwich ELISA to detect human F.IX in plasma after feeding (FIG. 6B).For the ELISA assay, 96 well flat-bottom ELISA plates from PIERCE(Rockford, Ill.) were coated with sheep-anti-human F.IX polyclonalantibody (Cedarlane Laboratories, Ontario, Canada), then blocked with10% BSA. Mouse plasma were diluted from {fraction (1/10)} to {fraction(1/10,000)} and added into the wells, incubated for two hours, washedwith wash buffer, and then HRP-conjugated polyclonal goat anti-humanF.IX antibodies (Cedarlane Laboratories) added for two hours. Color wasdeveloped by adding TMP, and results obtained using an ELISA platereader. As shown in FIG. 6B, the amount of F.IX in the blood of F.IX-fedmice was similar when measured by ELISA to that measured by the PBMC(FIG. 6A) and that most F.IX passes into the circulation as intactprotein.

[0128] To determine if fed human F.IX retains its native form andfunctional ability to treat bleeding episodes, F.IX-containingtransgenic pig milk (1.25 mg F.IX) was fed to hemophilia B mice (a F.IXknock-out obtained from Dr. Katherine High, Children's Hospital ofPhiladelphia; approximately 20 g in weight) over a period of two monthsevery other day and the effect on a bleeding defect determined. Bleedingtime was measured by cutting the tip of the tail and waiting for it toclot. Normal mice clot in about four minutes (FIG. 7A, open squares). Incontrast, hemophilia B mice do not stop bleeding (FIG. 7A, open circles)and the tail tip is cauterized to stop bleeding after fifteen minutes.As shown in FIG. 7A, oral administration of F.IX immediately correctedthe bleeding defect (FIG. 7A, closed circles). In addition, this resultis sustained for a period of at least two months (FIG. 7B), indicatingthat anti-F.IX antibodies are not being generated, which allows the F.IXto remain functional. Therefore, oral administration of human F.IX is aneffective treatment for bleeding, and does not induce inhibitoryimmunity, even following a long-term treatment.

[0129] Factor VIII

[0130] To demonstrate that hemophilia A, caused by the lack offunctional F.VIII, could also be treated by oral administration offactor, milk from a transgenic pig engineered to secrete hF.VIII was fedto F.VIII knock-out mice. However, this treatment had no effect on oraltolerance or on clotting times. It was possible that the dose of F.VIIIwas too low, since this particular transgenic strain of pig does notproduce large quantities of F.VIII (Paleyanda et al. Nature Biotech.15:971-5, 1997). Another explanation is that hF.VIII is almost 8 timeslarger than F.IX, with a molecular weight of nearly 240,000 kDa, whichmight make it difficult to pass into the bloodstream.

[0131] To distinguish between these possibilities, the studies wererepeated with commercially available F.VIII (Baxter) purified from humanplasma (Red Cross Holland labs, Rockville, Md.). hF.VIII (100 μg) wasadded to 0.5 ml of water or cow's milk and fed to hemophilia A mice bygavage. Bleeding time was assayed 45 minutes later. As shown in FIG. 8A,the bleeding time was corrected in hemophilia A mice fed hF.VIII eitherin water or milk. Therefore, sufficient hF.VIII passes into thebloodstream in native form to function as a clotting factor, despite thesize and complexity of the molecule.

[0132] To determine whether hF.VIII might be passing through smalllacerations created by the gavage process, F.VIII was fed to the mice ina more physiologic way, by dissolving 400 μg hF.VIII in 8 ml ofcommercial half cream/half milk and offering this to the mice in petridishes. Three mice drank about 12 ml of this mixture in about 2 hours,delivering about 200 μg to each mouse daily. Bleeding time was assayed45 minutes later. As shown in FIG. 8B, this method was also an effectivemethod to treat the bleeding disorder.

[0133] To demonstrate that feeding hF.VIII induces oral tolerance,hF.VIII was fed to hemophilia A mice (F.VIII knock-out mice) incommercial half and half (600 μg F.VIII/12 ml of half and half) dailyfor eight days. Control mice were fed plain half and half. On day 10,mice were bled to determine the bleeding time (FIG. 8C, pre-imm), thenimmunized with hF.VIII in CFA as described in the Examples above. Sevendays later, all mice were fed hF.V111-containing half-and-half todetermime if immunization-induced inhibitors would prevent the oral doseof F.VIII from functioning as a clotting factor.

[0134] A single immunization did not inhibit the function of the orallyadministered factor in previously orally tolerized mice (FIG. 8C, closedcircles), but did cause a loss of function in previously unfed controls(FIG. 8C, open circles). The test group was fed every other day,starting again from day 19 for an additional 7 days. Mice were thenboosted with hF.VIII in IFA, and again assayed for bleeding time 7 dayslater. Even after this second immunization with a strong adjuvant, thepreviously orally tolerized mice exhibited normal bleeding times, whilethe un-tolerized controls did not.

[0135] The mice were then sacrificed and checked for antibodies toF.VIII. Control mice had high levels of IgG2a antibodies (FIG. 8D, opencircles), which is consistent with their inability to respond totreatment with hF.VIII. In contrast, the orally tolerized mice hadbarely detectable levels of IgG2a (FIG. 8D, closed circles). Althoughorally tolerized mice did have some IgG1 antibodies specific forhF.VIII, after immunization (FIG. 8C), these antibodies behaved like thesimilar antibodies in orally tolerized wild type mice. These antibodiesare not inhibitors, as they did not affect the bleeding time (FIG. 8C).

[0136] This is the first demonstration that missing clotting factors canbe demonstrated in the circulation of hemophiliac mice as early as 45minutes after feeding, and that the orally-fed clotting factors arefunctional, as demonstrated by correction in bleeding time. Although itis commonly assumed that dietary proteins are digested completely tofree amino acids within the lumen of the gastrointestinal tract and thatonly trace amounts of macromolecular fragments enter the circulationhaving no clinical relevance, there is mounting evidence that suggeststhe opposite. The most compelling evidence showing that intact proteinsor macromolecular fragments are absorbed is provided by thedemonstration of antibodies to many food proteins that occur in thecirculation of healthy individuals and experimental animals. Forexample, 5% of recombinant human erythropoietin (about 40 kDa) fed to10-day old rats was demonstrated in the serum as early as one hour afterfeeding (Miller-Gilbert et al. 2001. Pediatr. Res. 50:261-7). Serumerythropoietin levels remained steady for up to 6 hours as opposed tothe rapid peak and fall observed after intravenous administration.Although orally administered erythropoietin showed functional activityin Hess mice by stimulation erythropoiesis, this could have been due tothe stimulation of erythropoietin receptors on the enterocytes withinthe gastrointestinal tract.

[0137] Similarly, gastrointestinal absorption of intact TGFβ1 wasdemonstrated after administration (by gavage) of radiolabeled TGFβ intothe stomachs of 5-day old pups (Letterio et al. 1994. Science264:1936-8). Although the neonatal gut is very permeable to intactproteins, similar results were observed in adult animals. In mice, rats,and fish fed ovalbumin, casein or HRP respectively, between 0.005 to0.7% of the total fed amount was measured in the circulation either byELlSA or Western analysis (Gonnella et al. 2001. J. Immunol.166:4456-64). Interestingly, the amount of antigen that passed into thecirculation was much higher in B-cell deficient mice compared to wildtype controls, suggesting that the lack of M cells does not affect thetransport of proteins across the gut.

[0138] Although both intravenous and oral administration of proteinsresults in detectable serum levels, antigen entry through the mucosaprovides certain benefits. First, pharmacokinetically, as opposed to arapid rise and decline as seen after intravenous administration, oraladministration provides relatively steady levels of the protein. Second,immunologically, it generates a gut-oriented immunity, which avoidsinhibitory or rejecting antibodies. Therefore, feeding F.VIII and F.IXin hemophilia A and B, respectively, is a simple, noninvasive andeffective method of treating hemophilia. In addition, it induces oraltolerance, avoiding the generation of inhibitory antibodies and avigorous T-cell response against missing clotting factors.

EXAMPLE 6 Comparison of Oral Administration of Clotting Factors in Waterand Milk

[0139] To determine if the presence of milk was critical to achieve oraltolerance and maintain proper function of the clotting factor, theadministration of F.VIII or F.IX in milk and water was compared.

[0140] Purified F.VIII from human serum was orally administered tohemophilia A mice (F.VIII knock out; Dr. Kang, U. of Iowa,) in cow'smilk or in water. Mice were fed 100 μg of F.VIII (antihemophilic factor,Baxter, Deerfield, Ill.) by gavage in 0.5 ml of water or cow's milk.Bleeding time was measured as described above, 45 minutes after feeding.As shown in FIG. 8A, feeding F.VIII derived from human plasma that isadded into cow's milk or water corrects bleeding time in hemophilia Amice. Therefore, the presence of milk when orally administering F.VIIIis not critical.

[0141] To determine if the presence of milk was critical to achieve oraltolerance to F.IX, normal B10.A mice were fed with low (10-20 μgF.IX/feed) or high (2500 μg F.IX/feed) does of human F.IX in pig milk oras purified plasma-derived human F.IX (Sigma) reconstituted in water(10-20 μg F.IX/feed) every other day for a total of five feeds. Controlmice were fed no F.IX. Two days later, mice were immunized with 0.5 μghuman F.IX emulsified in CFA at the base of the tail. Seven days later,the amount of T cell proliferation was determined using the methodsdescribed in EXAMPLE 1. As shown in FIGS. 9A and 9B, mice fed as littleas 20 μg F.IX in milk (FIG. 9A) were almost completely unresponsive,while T cells from mice fed with F.IX in water (FIG. 9B) retained asmall amount of activity (about 3% of unfed controls). However,administration of F.IX in water or milk resulted in better oraltolerance than no administration of F.IX.

[0142] In summary, a dose of 100 μg F.VIII/20 g body weight (about 5mg/kg), orally administered every day (or every other day), can be usedto increase hemostasis, thus treating hemophilia A. In addition, a doseof 1 mg F.IX/20 g body weight (about 50 mg/kg), orally administeredevery day (or every other day), is expected to increase hemostasis thustreating hemophilia B. However, it is possible that lower doses can beused, or that higher doses may be required in a particular subject, inorder to obtain a successful treatment of hemophilia.

EXAMPLE 7 Determination of Parameters for Low Dose Oral Tolerance

[0143] To identify optimal conditions to achieve low dose oral toleranceto human F.IX delivered in transgenic pig milk, the following methodscan be used. Similar methods can be used to determine the conditions forother antigens, including F.VIII, insulin, and protein C, in humans.

[0144] Lambs can be used as an experimental model. Sheep are an outbredspecies and, like humans (but unlike mice), their immune system becomesfunctional during the last trimester of pregnancy. They are also easierthan mice to bottle-feed.

[0145] The architecture of sheep intestines during the first 24-48 hoursof life is designed to collect maternal antibodies from colostrum, withvery high levels of Fc receptors that have a half-life of about 24hours. Therefore, feeding of F.IX milk is started 48 hours after birth,when the gut has settled.

[0146] Lambs are fed 0.3 mg F.IX/kg of animal/day, the amount of proteinshown to induce low dose tolerance in 20 g mice (FIGS. 1A-1C). Theeffective dose range in mice is 0.001 to 0.1 mg for OVA (0.05-5 mg/kg)and 0.003 to 0.1 mg/day for F.IX (0.15-0.5 mg/kg). Lambs are fed dailyfor three weeks with sheep milk supplemented with pig milk containinghuman F.IX (see EXAMPLE 5). A parallel control group of lambs issupplemented with normal pig milk containing no human F.IX. Milk iscollected, frozen immediately, and thawed just before use. Experimentswith mice demonstrate that continuous daily feeding is the mosteffective way of inducing oral tolerance.

[0147] Sheep can be immunized as early as two weeks of age. At threeweeks of age, half the lambs are immunized with F.IX in CFA after thefeedings are completed and then boosted two weeks later with F.IX inIFA. A dose of 100 μg human F.IX is sufficient to prime young ewes for asubsequent proliferative and antibody response. Lambs are tested forvarious types of immunity against F.IX after each immunization.

[0148] Three assays can be used to detect immune responses against F.IX.First, the amount of peripheral blood mononuclear cell proliferation canbe determined. One week and two weeks after immunization, 10-20 ml ofvenous blood is collected by jugular venipuncture, the mononuclear cellsfractionated using Percoll gradients and subsequently cultured in vitroin the presence of titrated amounts of F.IX, and assay for proliferationas described in EXAMPLES 1, 2 and 4.

[0149] Second, serum F.IX specific antibody titers can be determined.From each blood sample collected, serum is extracted. Dilutions of theserum are added to ELISA plates (Pierce) coated with human F.IX (10μg/ml). After a two hour incubation, the plates are washed with washbuffer, then incubated with polyclonal HRP-conjugated goat anti-humanF.IX antibodies (Cedarlane Laboratories) for two hours. For colordevelopment TMP is added and then the plates are read in an ELISA platereader. Antibodies of different classes can be identified using thesandwich ELISA method described in EXAMPLES 4 and 5.

[0150] Third, a factor IX inhibitor assay (Bethesda Assay) can beconducted. This functional assay measures the amount of inhibitorantibody in serum. The Bethesda assay is an in vitro functional assayperformed by mixing plasma containing unknown amounts of inhibitors(test plasma) with normal plasma to determine how the test plasmaprolongs the APTT of the normal plasma.

[0151] Lambs fed human F.IX should not generate a strong specificproliferative response, and should not develop antibodies against F.IX.If antibodies are produced, they will be of the secretory IgA type andwill not be inhibitory against F.IX.

[0152] It is possible that the determined effective dose in sheep orother subjects (such as humans) may differ from that in mice. If feeding0.3 mg/kg/day F.IX does not result in oral tolerance, experiments can berepeated using a titration of higher and lower doses of F.IX, todetermine the appropriate dose. In addition, it can also be determinedwhether feeding during the first 48 hours of life can enhance oraltolerance.

EXAMPLE 8 Determination of Parameters for High Dose Oral Tolerance

[0153] To determine parameters that are helpful to achieve optimal highdose oral tolerance to human F.IX delivered in transgenic pig milk, thefollowing methods can be used. Similar methods can be used to determinethe parameters for other antigens, including F.VIII, insulin, andprotein C.

[0154] Although low dose tolerance induces remarkable reductions incellular responses, it may allow for some antibody production. Thoughantibodies are unlikely to result in rejection of cells expressing aF.IX transgene, they may inhibit the secreted product. Therefore,tolerance can also be induced by feeding high doses of F.IX. Thisapproach has not been feasible in the past because of theextraordinarily high cost of purified F.IX. However, induction of oraltolerance does not require purified antigen. As experiments in sheepillustrate (FIGS. 5A-5C), newborns become tolerant of milk proteins evenwhen the proteins are given as mixtures in normal milk. The tolerizingability of unpurified transgenic pig milk containing 2.5 g/L F.IX (seeEXAMPLE 5) in the native situation where piglets feed continuously istested.

[0155] Non-transgenic piglets are fostered onto transgenic mothers andallowed to drink ad libitum until weaned. A control group of fosterlittermates is fostered onto non-transgenic control mothers. Two daysafter the animals are weaned, half of the piglets in each group aresubcutaneously immunized with F.IX (0.1 mg) in CFA and two weeks later,boosted with F.IX in IFA. Half of the piglets will be un-immunized ascontrols. One and two weeks after each immunization various classes ofimmunity will be determined as described above in EXAMPLE 7.

[0156] Piglets that drank F.IX containing milk from transgenic mothersare expected to be tolerant to FIX. This tolerance is tested bysubsequently immunizing these piglets to human F.IX in CFA as describedin the examples above. Tolerance is achieved if there is decreasedantibody formation and T cell proliferation in these piglets compared topiglets fostered onto a non-transgenic mother using the methodsdescribed in the examples above.

EXAMPLE 9 Oral Tolerance in Hemophiliac Puppies

[0157] This example describes procedures that can be used to developoral tolerance to clotting factors in hemophiliac puppies. The methodspecifically outlines methods for testing tolerance to F.IX inhemophiliac B puppies, but those skilled in the art will understand fromthe teachings herein that similar methods can be used to test fortolerance to F.VIII in hemophiliac A puppies, or for tolerance to anyantigen in any subject having an antigen-deficiency disease.

[0158] A canine model of hemophilia A is available (Giles et al. 1982.Blood 60:727-30; and Giles et al. 1984. Blood 63:451-6). Hemophilia Awas diagnosed in a miniature Schnauzer dog and inbreeding andcrossbreeding produced 16 hemophilic animals. Five animals developedpotent antibodies to canine F.VIII. In each case, the antibodiesrecognize both canine and human, but not porcine F.VII. Followinginhibitor development, infusion of canine cryoprecipitate washemostatically ineffective and F.VIII recovery at 30 minutes isnegligible.

[0159] Two canine models of hemophilia B are available. In the ChapelHill dog colony, hemophilia B is due to a missense mutation, andinhibitor formation in response to canine plasma or gene therapymaneuvers is relatively uncommon (Evans et al. 1989. Proc. Natl. Acad.Sci. U S A. 86:10095-9). However, for the Auburn dog colony, wheredisease is due to a small deletion that results in an early stop codon(Mauser et al. 1996. Blood. 88:3451-5), inhibitor formation is morefrequent. In both types of mutations, inhibitors to canine F.IX can beinduced by various means including repeated injection of purified canineF.IX, or injection of vector expressing canine F.IX. The rise ininhibitor antibodies correlates with fall of plasma F.IX levels (Herzoget al. 1999. Nat. Med. 5:56-63). Both colonies of dogs readily forminhibitory antibodies following infusion of human F.IX.

[0160] To assess the effect of oral tolerance on the development ofclotting factor inhibitors after immunization, canine models forhemophilia are used. F.IX is administered orally to normal puppies andto puppies with hemophilia B (Auburn dog colony) and their spontaneousimmune responses against F.IX analyzed.

[0161] The optimal feeding dose of F.IX found to prevent development ofinhibitory antibodies is used (see EXAMPLES 5-8). Normal and hemophiliaB puppies are divided into two groups: one group is fed F.IX-containingtransgenic pig milk, and the other group fed non-transgenic pig milk toserve as controls. Puppies are fed for six weeks until they are fullyweaned.

[0162] The puppies are then infused with 50 IU/kg human F.IXintravenously daily for two weeks, starting two days after weaning, orearlier if necessary to control bleeding. The generation of inhibitorantibodies after repeated intravenous administration of human F.IX hasbeen well established in the canine model (Evans et al. 1989. Proc.Natl. Acad. Sci. U S A. 86:10095-9). The course of the disease isfollowed, periodically assaying for inhibitory antibodies using theBethesda assay (see EXAMPLE 7), especially after F.IX infusions due tobleeding episodes.

[0163] PBMC proliferation and serum antibody titers against F.IX arealso compared between control and experimental animals, as described inEXAMPLE 4, except that the in vitro assays are modified to suit caninecells. For example, IMDM plus canine serum is a superior medium forcanine proliferation assays whereas RPM plus fetal calf serum serveswell for sheep.

[0164] It is expected that the intact antigen will gain access to thebloodstream after feeding, as was obtained for F.IX in mice (EXAMPLE 5).To test for F.IX in plasma samples from F.IX-fed hemophilia B and normalpuppies, methods described in EXAMPLE 5 are used.

[0165] To determine if the F.IX antigen remains functional after oraladministration, a functional assay is performed. For example, an APTTassay which measures the intrinsic coagulation activity of the plasmacan be used.

[0166] Therefore, in addition to orally tolerizing the immune system, itis determined whether small amounts of functional F.IX can reach theblood and help prevent bleeding episodes, thus increasing hemostasis.

[0167] It is expected that oral administration of F.IX by feeding willinduce oral tolerance and prevent the development of inhibitoryantibodies, as well as remain functional in the blood, and thereforeincrease hemostasis and serve as a treatment for hemophilia.

EXAMPLE 10 Oral Administration Followed by In Vivo Gene Expression

[0168] To demonstrate that administration and subsequent expression of aF.IX-containing transgene can be used to treat hemophilia,adeno-associated vectors containing a F.IX gene can be used.

[0169] After orally tolerizing puppies to F.IX as described in EXAMPLE9, adeno-associated viral vectors are used to introduce the human F.IXgene into hemophiliac puppies orally tolerized to human F.IX to increasehemostasis. Similar methods can be used in human hemophiliacs.

[0170] Methods for generating an introducing transgenes into a subjectare well known in the art. In addition, viral vectors for introducing aclotting factor into a subject are known. Examples of methods andvectors that can be used to introduce a clotting factor into a subject,include, but are not limited to those disclosed in: Stein et al., 2001.Mol. Ther. 3:850-6; Miller et al., 2001. N. Engl. J. Med. 344:1782-4;Emilien et al., 2000. Clin. Lab. Haematol. 22:313-23; Andrews et al.,2001. Mol. Ther. 3:329-36; Fabb et al., 2000. Curr. Opin. Mol Ther.2:601-6; and Lillicrap, 2000. Haematologica 85(10 Suppl): 108-12, aswell as other documents cited herein.

[0171] Following administration of the vector, F.IX levels in serum, aswell as immunity to F.IX, are monitored as described in EXAMPLES 5 and7, over a period of one year or until the factor disappears, whichevercomes first.

[0172] It is expected that oral administration of a clotting factor byfeeding will induce oral tolerance and prevent the development ofinhibitory antibodies, such that when the factor is subsequentlyexpressed recombinantly in the subject, the recombinant protein will notbe inactivated by anti-clotting factor antibodies. This will result infunctional clotting factor in the blood, and therefore increasehemostasis and serve as a treatment for hemophilia.

EXAMPLE 11 Oral Tolerance and Treatment in Humans

[0173] This example describes methods that can be used to inducetolerance to F.IX in a human hemophiliac. Similar methods can be used toinduce oral tolerance to any antigen of interest in a human subject, forexample F.VIII, insulin and protein C.

[0174] Among hemophilia patients that form inhibitors, the incidence ofpatients with large deletions is known (deletion/total inhibitorproduction=0.5). However, due to the cost of widespread geneticscreening, and the lack of practical consequence to the management ofhemophilia patients, the incidence of inhibitor producers among thosewith large deletions is not known (inhibitor production/largedeletions).

[0175] The patient group most likely to benefit from treatment arechildren that have no F.IX, thus making them particularly prone tomaking inhibitors. After determining the F.IX levels in plasma of ahemophiliac child, patients having undetectable levels are furthertested to determine whether they have a large F.IX deletion. From thisstudy, the incidence of inhibitor producers among those who have largedeletions can be determined.

[0176] From studies on hemophiliac puppies, patients with largedeletions are believed to be particularly prone to making inhibitors andthus particularly at risk of becoming refractory to replacement therapy.

[0177] Hemophiliac children are orally tolerized by oral administration(feeding) of F.IX-containing transgenic milk. Alternatively, purifiedF.IX can be administered orally in any medium suitable for oraladministration, including water.

[0178] The immune response to F.IX during the course of the disease ismonitored as described in EXAMPLE 7. In addition, the amount offunctional F.IX in the blood, the formation of antibodies against F.IXand PBMC proliferation are monitored as described above.

[0179] In addition, hemostasis is monitored, using an APTT assaydescribed in EXAMPLE 7 or a bleeding time assay. Treatment is successfulif hemostasis is increased relative to hemostasis prior to oraladministration of the clotting factor, or relative to untreatedhemophiliacs.

EXAMPLE 12 Oral Tolerance Followed by In Vivo Gene Expression

[0180] As an alternative to continually orally administering a clottingfactor to a subject to increase hemostasis, once oral tolerance isachieved, long term treatment of the clotting disorder can be achievedby expressing the deficient clotting factor in vivo. Similar methods canbe used to treat any antigen-deficiency disease.

[0181] The present disclosure provides methods of expressing a proteinin a cell or tissue in vivo. In one embodiment, transfection of the cellor tissue occurs in vitro. In this example, the cell or tissue (such asa graft) is removed from a subject and then transfected with anexpression vector containing a cDNA encoding the protein of interest.The transfected cells will produce functional protein and can bereintroduced into the subject. In another embodiment, a nucleic acidencoding the protein of interest is administered to a subject directly,and transfection occurs in vivo.

[0182] The methods disclosed herein can be used to treating a subjectwith an antigen-deficiency disease such as a subject having hemophilia Aor B. Such a method would increase hemostasis (decrease blood clottingtime) in hemophiliacs or subjects having other defects in bloodclotting.

[0183] The scientific and medical procedures required for human celltransfection are now routine. The public availability of numerousprotein and cDNA sequences allows the development of human (and othermammals) in vivo gene expression based upon these procedures.Immunotherapy of melanoma patients using genetically engineeredtumor-infiltrating lymphocytes (TILs) has been reported by Rosenberg etal. (N. Engl. J. Med. 323:570-8, 1990). In that study, a retrovirusvector was used to introduce a gene for neomycin resistance into TILS. Asimilar approach may be used to introduce a clotting factor cDNA intohemophiliacs.

[0184] In some embodiments, a method of treating subjects which underexpress a functional protein, or in which greater functional proteinexpression is desired, is disclosed. These methods can be accomplishedby introducing a gene coding for the desired protein into a subject. Ageneral strategy for transferring genes into donor cells is disclosed inU.S. Pat. No. 5,529,774, incorporated by reference. Generally, a geneencoding a protein having therapeutically desired effects is cloned intoa viral expression vector, and that vector is then introduced into thetarget organism. The virus infects the cells, and produces the proteinsequence in vivo, where it has its desired therapeutic effect. Zabner etal. (Cell 75:207-16, 1993).

[0185] It may only be necessary to introduce the genetic or proteinelements into certain cells or tissues. However, in some instances, itmay be more therapeutically effective and simple to treat all of asubject's cells, or more broadly disseminate the vector, for example byintravascular (i.v.) administration.

[0186] The nucleic acid sequence encoding at least one therapeuticagent, such as a clotting factor, is under the control of a suitablepromoter. Suitable promoters which may be employed include, but are notlimited to, the gene's native promoter, retroviral LTR promoter, oradenoviral promoters, such as the adenoviral major late promoter; theCMV promoter; the RSV promoter; inducible promoters, such as the MMTVpromoter; the metallothionein promoter; heat shock promoters; thealbumin promoter; the histone promoter; the α-actin promoter; TKpromoters; B19 parvovirus promoters; and the ApoAI promoter. However thescope of the disclosure is not limited to specific foreign genes orpromoters.

[0187] The recombinant nucleic acid can be administered to the subjectby any method which allows the recombinant nucleic acid to reach theappropriate cells. These methods include injection, infusion,deposition, implantation, or topical administration. Injections can beintradermal or subcutaneous. The recombinant nucleic acid can bedelivered as part of a viral vector, such as avipox viruses, recombinantvaccinia virus, replication-deficient adenovirus strains or poliovirus,or as a non-infectious form such as naked DNA or liposome encapsulatedDNA, as further described in EXAMPLE 13.

EXAMPLE 13 Viral Vectors for In Vivo Gene Expression

[0188] Adenoviral vectors include essentially the complete adenoviralgenome (Shenk et al., Curr. Top. Microbiol. Immunol. 111:1-39, 1984).Alternatively, the adenoviral vector is a modified adenoviral vector inwhich at least a portion of the adenoviral genome has been deleted. Inone embodiment, the vector includes an adenoviral 5′ ITR; an adenoviral3′ ITR; an adenoviral encapsidation signal; a DNA sequence encoding atherapeutic agent; and a promoter for expressing the DNA sequenceencoding a therapeutic agent. The vector is free of at least themajority of adenoviral E1 and E3 DNA sequences, but is not necessarilyfree of all of the E2 and E4 DNA sequences, and DNA sequences encodingadenoviral proteins transcribed by the adenoviral major late promoter.In another embodiment, the vector is an adeno-associated virus (AAV)such as described in U.S. Pat. No. 4,797,368 (Carter et al.) and inMcLaughlin et al. (J. Virol. 62:1963-73, 1988) and AAV type 4 (Chioriniet al. J. Virol. 71:6823-33, 1997) and AAV type 5 (Chiorini et al. J.Virol. 73:1309-19, 1999)

[0189] Such a vector can be constructed according to standardtechniques, using a shuttle plasmid which contains, beginning at the 5′end, an adenoviral 5′ ITR, an adenoviral encapsidation signal, and anE1a enhancer sequence; a promoter (which may be an adenoviral promoteror a foreign promoter); a tripartite leader sequence, a multiple cloningsite (which may be as herein described); a poly A signal; and a DNAsegment which corresponds to a segment of the adenoviral genome. The DNAsegment serves as a substrate for homologous recombination with amodified or mutated adenovirus, and may encompass, for example, asegment of the adenovirus 5′ genome no longer than from base 3329 tobase 6246. The plasmid can also include a selectable marker and anorigin of replication. The origin of replication may be a bacterialorigin of replication. A desired DNA sequence encoding a therapeuticagent can be inserted into the multiple cloning site of the plasmid.

[0190] Examples of vectors which can be used to practice the methodsdisclosed herein include those disclosed in: WO 95/27512 to Woo et al.;WO 127303 to Walsh et al.; U.S. Pat. No. 6,221,349 to Couto et al.; U.S.Pat. No. 6,093,392 to High et al.

EXAMPLE 14 Production of Sequence Variants

[0191] Disclosed herein methods for treating antigen-deficiency diseasesby oral administration of the appropriate antigen or protein. It isunderstood by those skilled in the art that use of non-native antigensequences (such as polymorphisms, fragments, or variants) can be used topractice the methods of the present disclosure, as long as thedistinctive functional characteristics of the antigen are retained. Forexample, F.IX or F.VIII variants can be used to practice the methodsdisclosed herein if they retain their ability to increase hemostasis.This activity can readily be determined using the assays disclosedherein, for example those described in EXAMPLES 5 and 9. In yet otherembodiments, F.IX or F.VIII has the distinct characteristic of beingreduced in patients suffering from hemophilia.

[0192] This disclosure facilitates the use of DNA molecules, and therebyproteins, derived from a native protein but which vary in their precisenucleotide or amino acid sequence from the native sequence. Suchvariants can be obtained through standard molecular biology laboratorytechniques and the sequence information disclosed herein.

[0193] DNA molecules and nucleotide sequences derived from a native DNAmolecule can also be defined as DNA sequences which hybridize understringent conditions to the DNA sequences disclosed, or fragmentsthereof. Hybridization conditions resulting in particular degrees ofstringency vary depending upon the nature of the hybridization methodand the composition and length of the hybridizing DNA used. Generally,the temperature of hybridization and the ionic strength (especially theNa⁺ concentration) of the hybridization buffer determines hybridizationstringency. Calculations regarding hybridization conditions required forattaining particular amounts of stringency are discussed by Sambrook etal. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989, Chapters 9 and 11), herein incorporated by reference.Hybridization with a target probe labeled with [³²P]-dCTP is generallycarried out in a solution of high ionic strength such as 6×SSC at atemperature that is about 5-25° C. below the melting temperature, T_(m).An example of stringent conditions is a salt concentration of at leastabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and a temperature of at least about 30° C. for short probes (e.g. 10to 50 nucleotides). Stringent conditions can also be achieved with theaddition of destabilizing agents such as formamide. For example,conditions of 5× SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH7.4) at 25-30° C. are suitable for allele-specific probe hybridizations.

[0194] The degeneracy of the genetic code further widens the scope ofthe present disclosure as it enables major variations in the nucleotidesequence of a DNA molecule while maintaining the amino acid sequence ofthe encoded protein. For example, the amino acid Ala is encoded by thenucleotide codon triplet GCT, GCG, GCC and GCA. Thus, the nucleotidesequence could be changed without affecting the amino acid compositionof the encoded protein or the characteristics of the protein. Based uponthe degeneracy of the genetic code, variant DNA molecules may be derivedfrom a cDNA molecule using standard DNA mutagenesis techniques asdescribed above, or by synthesis of DNA sequences. DNA sequences whichdo not hybridize under stringent conditions to the cDNA sequencesdisclosed by virtue of sequence variation based on the degeneracy of thegenetic code are also comprehended by this disclosure.

[0195] Clotting factor variants, fragments, fusions, and polymorphismswill retain the ability to increase hemostasis, as determined using theassays disclosed herein, for example by performing an APTT assay(EXAMPLE 9) or a bleeding time assay. Variants and fragments of aprotein may retain at least 70%, 80%, 85%, 90%, 95%, 98%, or greatersequence identity to a protein amino acid sequence and maintain thefunctional activity of the protein as understood by those in skilled inthe art. Examples of clotting factor variants, include, but are notlimited to those disclosed in U.S. Pat. No. 6,221,349 to Couto et al.;U.S. Pat. No. 4,877,614 to Andersson et al.; EP 770396 to Zimmermann etal.; U.S. Pat. No. 6,093,392 to High et al.; and EP 0294910 to van Ooyenet al.

EXAMPLE 15 Recombinant Expression of Proteins

[0196] With publicly available cDNA and corresponding amino acidsequences, as well as the disclosure herein of variants, fragments andfusions thereof, the expression and purification of any publicly knownprotein by standard laboratory techniques is enabled. The purifiedprotein can be used for patient therapy. For example, mammals thatproduce recombinant F.VIII or F.IX in their milk are known (see U.S.Pat. No. 5,880,327 to Lubon et al. and Van Cott et al. 1999. Genet.Anal. 15:155-60, respectively), and can be used to practice the methodsdisclosed herein. However, one skilled in the art will understand thatthe orally administered clotting factor can be produced in any cell ororganism of interest, and purified prior to administration to a subject,as an alternative to feeding the subject milk containing the recombinantclotting factor.

[0197] Methods for producing recombinant proteins are well known in theart. Therefore, the scope of this disclosure includes recombinantexpression of any antigen/protein. For example, see U.S. Pat. No.5,342,764 to Johnson et al.; U.S. Pat. No. 5,846,819 to Pausch et al.;U.S. Pat. No. 5,876,969 to Fleer et al. and Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 17,herein incorporated by reference).

[0198] In addition, methods for producing recombinant clotting factorsare known, for example as disclosed in EP 0294910 (and referencestherein); EP 0160457; EP 0253455; EP 0150735; U.S. Pat. No. 6,221,349 toCouto et al.; U.S. Pat. No. 5,804,420 to Chan et al. and U.S. Pat. No.4,770,999 to Kaufman et al.

EXAMPLE 16 Peptide Modifications

[0199] Orally administered proteins can be modified using a variety ofchemical techniques to produce derivatives having essentially the sameactivity as the unmodified peptides, and optionally having otherdesirable properties. For example, carboxylic acid groups of thepeptide, whether carboxyl-terminal or side chain, can be provided in theform of a salt of a pharmaceutically-acceptable cation or esterified toform a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ whereinR₁ and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to forma heterocyclic ring, such as a 5- or 6- membered ring. Amino groups ofthe peptide, whether amino-terminal or side chain, can be in the form ofa pharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

[0200] Hydroxyl groups of the peptide side chain can be converted toC₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques.Phenyl and phenolic rings of the peptide side chain can be substitutedwith one or more halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof, or amides ofsuch carboxylic acids. Methylene groups of the peptide side chains canbe extended to homologous C₂-C₄ alkylenes. Thiols can be protected withany one of a number of well-recognized protecting groups, such asacetamide groups. Those skilled in the art will also recognize methodsfor introducing cyclic structures into the peptides disclosed herein toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a carboxyl-terminal oramino-terminal cysteine residue can be added to the peptide, so thatwhen oxidized the peptide will contain a disulfide bond, generating acyclic peptide. Other peptide cyclizing methods include the formation ofthioethers and carboxyl- and amino-terminal amides and esters.

[0201] To maintain a functional peptide, particular peptide variantswill differ by only a small number of amino acids from a peptide. Suchvariants can have deletions (for example of 1-3 or more amino acids),insertions (for example of 1-3 or more residues), or substitutions thatdo not interfere with the desired activity of the peptide.Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. In particular embodiments, such variants have amino acidsubstitutions of single residues, for example 1, 3, 5 or even 10substitutions in a protein.

[0202] Peptidomimetic and organomimetic embodiments are also disclosedherein, whereby the three-dimensional arrangement of the chemicalconstituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid sidechains in the peptide, resulting in such peptido- andorganomimetics of a clotting factor having the ability to increasehemostasis. For computer modeling applications, a pharmacophore is anidealized, three-dimensional definition of the structural requirementsfor biological activity. Peptido- and organomimetics can be designed tofit each pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology (ed. Munson, 1995), chapter 102 for adescription of techniques used in CADD.

EXAMPLE 17 Pharmaceutical Compositions and Modes of Administration

[0203] The pharmaceutically effective carriers useful herein areconventional. Remington's Pharmaceutical Sciences, by Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery.

[0204] Oral Administration of Antigens and Proteins

[0205] In an embodiment in which an antigen, such as a clotting factor,is administered to a subject, the antigen is delivered enterally, forexample orally (e.g., by feeding), or rectally (e.g., by suppository).The present disclosure also provides pharmaceutical compositions whichinclude a therapeutically effective amount of a clotting factor alone orwith a pharmaceutically acceptable carrier. Furthermore, thepharmaceutical compositions or methods of treatment can be administeredin combination (or separately) with one or more other therapeutictreatments, such as other agents that increase hemotasis. Embodiments ofthe disclosure comprising medicaments can be prepared with conventionalpharmaceutically acceptable carriers, adjuvants and counterions as wouldbe known to those of skill in the art.

[0206] The antigen can be administered in combination with at least one,for example one or more pharmaceutically effective carriers, such as apharmaceutically and physiologically acceptable fluid. Examples ofpharmaceutically effective carriers include, but are not limited tomilk, water, physiological saline, balanced salt solutions, aqueousdextrose, sesame oil, glycerol, ethanol, combinations thereof, or thelike, as a vehicle. The carrier and composition can be sterile, and theformulation suits the mode of administration. In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

[0207] The composition can be a liquid solution, suspension, emulsion,tablet, pill, capsule, sustained release formulation, or powder, but nota liposome also containing a protease inhibitor. For solid compositions(e.g., powder, pill, tablet, or capsule forms), conventional non-toxicsolid carriers can include, for example, pharmaceutical grades ofmannitol, lactose, starch, sodium saccharine, cellulose, magnesiumcarbonate, or magnesium stearate. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides.

[0208] The amount of antigen, such as a clotting factor, effective inthe treatment of a particular disorder or condition, such as hemophilia,will depend on the nature of the disorder or condition, and can bedetermined by standard clinical techniques. In addition, in vitro assayscan be employed to identify optimal dosage ranges (see EXAMPLES 7 and8). The precise dose to be employed in the formulation will also dependon the seriousness of the disease or disorder, and should be decidedaccording to the judgment of the practitioner and each subject'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

[0209] The disclosure also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.Instructions for use of the composition can also be included.

[0210] Administration of Nucleic Acid Molecules

[0211] In an embodiment in which a nucleic acid is employed to allowexpression of a nucleic acid in a cell, the nucleic acid is deliveredintracellularly (e.g., by expression from a nucleic acid vector or byreceptor-mediated mechanisms). In one embodiment, a nucleic acid encodesfor an antigen, such as a clotting factor.

[0212] Various delivery systems for administering a nucleic acid areknown, and include encapsulation in liposomes, microparticles,microcapsules, receptor-mediated endocytosis (Wu and Wu, J. Biol. Chem.1987, 262:4429-32), and construction of therapeutic nucleic acids aspart of a retroviral or other vector. Methods of introduction include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, and oral routes. The compoundsmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal, vaginal and intestinal mucosa, etc.)and may be administered together with other biologically active agents.Administration can be systemic or local. Pharmaceutical compositions canbe introduced into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

[0213] Liposomes fuse with the target site and deliver the contents ofthe lumen intracellularly. The liposomes are maintained in contact withthe target cells for a sufficient time for fusion to occur, usingvarious means to maintain contact, such as isolation and binding agents.Liposomes can be prepared with purified proteins or peptides thatmediate fusion of membranes, such as Sendai virus or influenza virus.The lipids may be any useful combination of known liposome forminglipids, including cationic lipids, such as phosphatidylcholine. Otherpotential lipids include neutral lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like. For preparingthe liposomes, the procedure described by Kato et al. (J. Biol. Chem.1991, 266:3361) can be used.

[0214] Where the therapeutic molecule is a nucleic acid, administrationcan be achieved by an appropriate nucleic acid expression vector whichis administered so that it becomes intracellular, e.g., by use of aretroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al., Proc. Natl. Acad. Sci. USA 1991, 88:1864-8), etc.Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

[0215] The vector pcDNA, is an example of a method of introducing theforeign cDNA into a cell under the control of a strong viral promoter(CMV) to drive the expression. However, other vectors can be used (seeEXAMPLES 13 and 15). Other retroviral vectors (such as pRETRO-ON,Clontech), also use this promoter but have the advantages of enteringcells without any transfection aid, integrating into the genome oftarget cells only when the target cell is dividing (as cancer cells do,especially during first remissions after chemotherapy) and they areregulated. It is also possible to turn on the expression of a nucleicacid by administering tetracycline when these plasmids are used.

[0216] Other plasmid vectors, such as pMAM-neo (Clontech) or pMSG(Pharmacia) use the MMTV-LTR promoter (which can be regulated withsteroids) or the SV10 late promoter (pSVL, Pharmacia) ormetallothionein-responsive promoter (pBPV, Pharmacia) and other viralvectors, including retroviruses. Examples of other viral vectors includeadenovirus, AAV (adeno-associated virus), recombinant HSV, poxviruses(vaccinia) and recombinant lentivirus (such as HIV). These vectorsachieve the basic goal of delivering into the target cell the cDNAsequence and control elements needed for transcription. The presentdisclosure includes all forms of nucleic acid delivery, includingsynthetic oligos, naked DNA, plasmid and viral, integrated into thegenome or not.

[0217] Having illustrated and described methods for treating hemophiliaA or B by oral administration of a therapeutically effective amount ofF.VIII or F.IX, respectively, it should be apparent to one skilled inthe art that the disclosure can be modified in arrangement and detailwithout departing from such principles. In view of the many possibleembodiments to which the principles of our disclosure may be applied, itshould be recognized that the illustrated embodiments are onlyparticular examples of the disclosure and should not be taken as alimitation on the scope of the disclosure. Rather, the scope of thedisclosure is in accord with the following claims. We therefore claim asour invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A method for increasing hemostasis in a subject having ahemophilia, comprising orally administering to the subject atherapeutically effective amount of a clotting factor sufficient toinduce oral tolerance and supply exogenous clotting factor to thesubject, wherein the clotting factor is not present in a liposome. 2.The method of claim 1, wherein the hemophilia is a result of impairedexpression of the clotting factor, and the clotting factor isadministered as long as the hemophilia persists.
 3. The method of claim1, wherein increasing hemostasis comprises decreasing blood clottingtime.
 4. The method of claim 1, wherein the clotting factor isadministered in absence of an anti-immune therapy.
 5. The method ofclaim 1, wherein the therapeutically effective amount of a clottingfactor is at least 5 mg of clotting factor per 1 kg of subject daily. 6.The method of claim 1, wherein the therapeutically effective amount of aclotting factor is at least 50 mg of clotting factor per 1 kg of subjectdaily.
 7. The method of claim 1, wherein the subject is a mammal.
 8. Themethod of claim 7, wherein the mammal is a human.
 9. The method of claim8, wherein the hemophilia is hemophilia A and the clotting factor isfactor VIII (F.VIII).
 10. The method of claim 8, wherein the hemophiliais hemophilia B and the clotting factor is factor IX (F.IX).
 11. Themethod of claim 9, wherein the therapeutically effective amount ofF.VIII is at least 5 mg of clotting factor per 1 kg of subject daily.12. The method of claim 10, wherein the therapeutically effective amountof F.IX is at least 50 mg of clotting factor per 1 kg of subject daily.13. The method of claim 10, wherein the F.IX is present in milk obtainedfrom a transgenic mammal expressing recombinant F.IX.
 14. The method ofclaim 13, wherein the mammal is a pig.
 15. The method of claim 3,wherein the blood clotting time is measured using an activated partialthromboplastin time (APTT) assay.
 16. The method of claim 1, whereinoral administration is achieved by feeding the subject a therapeuticallyeffective amount of the clotting factor.
 17. The method of claim 16,wherein the clotting factor is a recombinant clotting factor present inmilk of a transgenic mammal.
 18. The method of claim 1, wherein theclotting factor is present in a pharmaceutically effective carrier. 19.The method of claim 18, wherein the pharmaceutically effective carrieris water.
 20. The method of claim 18, wherein the pharmaceuticallyeffective carrier is milk.
 21. The method of claim 1, further comprisingadministering to the subject an isolated recombinant nucleic acidmolecule encoding the clotting factor, wherein the clotting factor isexpressed in the subject.
 22. A method for increasing hemostasis in asubject having a hemophilia, consisting of orally administering to thesubject a therapeutically effective amount of a clotting factor and atleast one pharmaceutically acceptable carrier which is not a liposome.23. A method for orally administering a recombinant clotting factor to asubject for treatment of a hemophilia comprising orally administering amilk containing recombinant clotting factor to the subject at atherapeutically effective amount such that hemostasis in the subject isincreased, thereby treating the hemophilia.
 24. The method of claim 23,wherein the milk comprises at least 2 g recombinant clotting factor perliter of milk.
 25. The method of claim 24, wherein the milk is obtainedfrom a transgenic mammal.
 26. The method of claim 25, wherein the mammalis a pig.
 27. The method of claim 26, wherein the milk comprisesrecombinant F.IX antigen at a concentration of about 2 g per liter. 28.A method for orally administering a recombinant clotting factor to asubject for treatment of a hemophilia comprising: expressing therecombinant clotting factor in a mammal such that the clotting factor isexpressed in milk of the mammal; and orally administering the milk inwhich the clotting factor has been expressed to the subject at atherapeutically effective amount such that blood clotting time in thesubject is reduced, thereby treating the hemophilia.
 29. The method ofclaim 28, wherein the hemophilia is hemophilia B and the clotting factoris F.IX.
 30. A method for orally administering a recombinant F.IXprotein to a subject for treatment of hemophilia B comprising orallyadministering milk containing recombinant F.IX protein to the subject ata therapeutically effective amount such that blood clotting time in thesubject is reduced, thereby treating the hemophilia B.
 31. A method oforally administering to a hemophiliac a therapeutically effective amountof a clotting factor protein sufficient to induce oral tolerance to theclotting factor protein and supply exogenous clotting factor protein tothe hemophiliac, wherein the clotting factor protein is not present in aliposome, and wherein the oral tolerance prepares the hemophiliac for invivo expression of a clotting factor gene.
 32. The method of claim 31,wherein the clotting factor gene is a F.VIII gene and the clottingfactor protein is a F.VIII protein.
 33. The method of claim 31, whereinthe clotting factor gene is a F.IX gene and the clotting factor proteinis a F.IX protein.